The present invention relates to toxin conjugates in which a toxin is bound through a spacer to a residue derived from a compound which has an affinity for a target cell, for example, a residue derived from an antibody or antibody fragment which is specific to a cancer. The toxin conjugate obtained by the present invention inhibits the growth of a target cell selectively and efficiently, and is useful as an active ingredient of an antitumor agent.
Anthracycline anticancer compounds so far known include daunomycin (U.S. Pat. No. 3,590,028) and adriamycin (U.S. Pat. No. 3,590,028), which are in wide clinical use as anticancer agents. However, side effects of these compounds have been reported; for example, adriamycin is known to have side effects such as cardial toxicity and marrow depression [Cancer Chemotherapy and Pharmacology, 4, 5-10 (1980)]. Alleviation of such side effects is a big problem to be solved, and comprehensive research has so far been made to this end. Specifically, in recent years, research on drug delivery systems has been pursued aiming at alleviation of toxicity, maintenance of concentration in blood and improvement of affinity for a cancer cell. For example, the modification with a copolymer of divinyl ether-maleic anhydride (Japanese Published Unexamined Patent Application No. 67490/85), and the modification with dextran [Cancer Treatment Reports, 66, 107 (1982)] have been reported.
Further, antibody conjugates (toxin conjugates) having a specificity to a cancer cell have been studied. Some examples of such conjugates are shown below [Bioconjugate Chem., 1, 13 (1990)].
There are some other reports relating to antibody conjugates [Japanese Published Unexamined Patent Application No. 67433/85; Japanese Published Unexamined Patent Application No. 35575/88; Japanese Published Unexamined Patent Application No. 150282/88; Japanese Published Unexamined Patent Application No. 246336/88; Biochem. J., 173, 723 (1978); Cancer Res., 50, 6600 (1990); Science, 261, 212 (1993); Bioconjugate Chem., 4, 275 (1993); Bioconjugate Chem., 4, 251 (1993); Bioconjugate Chem., 5, 88 (1994); Bioconjugate Chem., 5, 31 (1994); and Bioconjugate Chem., 5, 246 (1994)].
There are also known examples in which low molecular weight polyethylene glycol is used as a spacer [Proc. Natl. Acad. Sci. USA, 88, 9287 (1991); PCT National Publication No. 508856/93; and Bioconjugate Chem., 4, 455 (1993)], and examples of the modification of an antibody with polyethylene glycol (WO 93/08838 and WO 86/04145). Further, the use of a spacer containing a peptide has been reported [U.S. Pat. No. 4,671,958; PCT National Publication No. 502886/93; and Bioconjugate Chem., 4, 10 (1993)].
The present inventors made intensive studies in search of an excellent toxin conjugate which kills tumor cells selectively. As a result, the inventors have found that a conjugate having a spacer which is specifically cleaved when introduced into a specific cell can be obtained by chemically binding a toxin to a compound which has a specific affinity for a cancer cell through a novel spacer comprising polyethylene glycol and dipeptide. Thus the present invention has been completed.
The present invention relates to a toxin conjugate in which a residue derived from a compound having an affinity for a target cell is bound to a toxin through a spacer comprising polyalkylene glycol and dipeptide.
Typical examples of the conjugates of the present invention are toxin conjugates represented by general formula (A):
Z"Parenopenst"X1xe2x80x94CH2(OCH2CH2)nOCH2COxe2x80x94R1xe2x80x94R2xe2x80x94Wxe2x80x94Y1)mxe2x80x83xe2x80x83(A)
wherein Z represents a residue derived from a compound having an affinity for a target cell; Y1 represents a toxin; R1 and R2, which may be the same or different, each represents an amino acid residue; Alk represents alkylene; n represents an integer of 1-1000; and m represents an integer of 1-100. Although X0, W0 and W1 are not specifically defined, examples of their representations are as follows: X0 represents xe2x80x94COAlk1-, xe2x80x94SAlk1-, xe2x80x94COOAlk1-, xe2x80x94CONHAlk1-, xe2x80x94COAlk1COxe2x80x94, 
W0 represents CO, -Alk1COxe2x80x94, or -Alk1Sxe2x80x94; and W1 represents a single bond, S, xe2x80x94OAlk1COxe2x80x94, xe2x80x94NHAlk1COxe2x80x94, xe2x80x94NHAlk1NHxe2x80x94, 
In the above formulae, Alk1 and Alk2, which may be the same or different, each represents a straight-chain or branched alkylene having 1-8 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, and octylene.
Particularly, preferred toxin conjugates are compounds represented by general formula (I):
Z"Parenopenst"X1xe2x80x94CH2(OCH2CH2)nOCH2COxe2x80x94R1xe2x80x94R2xe2x80x94Wxe2x80x94Y1)mxe2x80x83xe2x80x83(I)
wherein X1 represents CO, S or 
W represents a single bond or 
and Z, Y1, R1, R2, n and m have the same meanings as defined above. The compounds represented by general formula (I) are hereinafter referred to as Compounds (I), and the same applies to the compounds of other formula numbers.
In the definitions of the above-described groups, the alkylene moiety of the alkylene and the polyalkylene glycol means a straight-chain or branched alkylene having 1-8 carbon atoms such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, hexylene, heptylene, and octylene. Examples of the compounds which have an affinity for a target cell are compounds having a structure capable of binding to X1 such as COOH, NH, SH, and OH, e.g., receptor ligands such as epidermal growth factors (EGF) and transferrin having an affinity for a target cell, adhesion molecules represented by the arginine-glycine-aspartic acid sequence, and proteins and peptides such as antibodies and antibody fragments. Preferred examples are antibodies and antibody fragments. The antibodies include polyclonal antibodies and monoclonal antibodies produced according to known methods which belong to immunoglobulin (Ig) classes such as IgG, IgA, IgM, and IgE, and immunoglobulin subclasses, for example, IgG1, IgG2, IgG3, and IgG4 in the case of IgG. Preferred examples are KM-641 antibody which is an antibody against ganglioside GD3 which is highly expressed in a cancer cell (Japanese Published Unexamined Patent Application No. 176791/93), KM-231 (AMC-462) antibody which is an antibody against sialyl Lewis a (Japanese Published Unexamined Patent Application No. 021562/88), and NL-1 antibody which is an antibody against common human acute lymphatic leukemia cell antigen (CALLA) [Proc. Natl. Acad. Sci. USA 79, 4386-4390 (1982)]. Examples of the antibody fragments are F(abxe2x80x2)2 obtained by treating the above-mentioned antibodies with a proteolytic enzyme such as pepsin, Fabxe2x80x2 obtained by reducing F(abxe2x80x2)2 with mercaptan, and Fab obtained by degrading the antibodies with a proteolytic enzyme such as papain, trypsin, chymotrypsin, and plasmin. F(abxe2x80x2)2, Fabxe2x80x2, and Fab are known as well as methods for producing them [Immunochemistry, Yuichi Yamamura et al., p. 461, Asakura Shoten (1973)]. Examples of the toxins are toxins having a structure capable of condensing with a carboxyl group of the terminal amino acid R2 or capable of attaching to a double bond of maleinimide, such as NH, SH and OH, e.g., anthracycline compounds such as adriamycin (U.S. Pat. No. 3,590,028) and daunorubicin (U.S. Pat. No. 3,616,242), duocarmycin derivatives such as DC-88A derivatives (Japanese Published Unexamined Patent Application No. 288879/90) and the compounds described in Reference Examples, mitomycin A, mitomycin C, and protein toxins such as ricin A, diphtheria toxin, and Pseudomonas exotoxin. Examples of the amino acid residues are an alanine residue, a leucine residue, a glycine residue, a proline residue and a valine residue.
The abbreviations used herein have the following meanings, unless otherwise specified.
The abbreviations for amino acids and their protecting groups follow the recommendations by IUPAC-IUB Joint Commission on Biochemical Nomenclature [Biochemistry, 11, 1726 (1972)].
Ala: L-Alanine
Val: L-Valine
Pro: L-Proline
Gly: Glycine
DMF: N,N-Dimethylformamide
DMSO: Dimethylsulfoxide
THF: Tetrahydrofuran
TFA: Trifluoroacetic acid
NMM: N-Methylmorpholine
Bzl: Benzyl
tBu: tert-Butyl
Z: Benzyloxycarbonyl
Pic: Picolyl
HONSu: N-Hydroxysuccinimide
ONSu: Succinimidoxy
DCC: N,Nxe2x80x2-Dicyclohexylcarbodiimide
DCU: N,Nxe2x80x2-Dicyclohexylurea
ADM: Adriamycin
DNR: Daunorubicin
HOBt: N-Hydroxybenzotriazole
PyBOP: Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
DMAP: 4-(N,N-Dimethylamino) pyridine
PEG: COCH2(OCH2CH2)nOCH2CO
HPLC: High performance liquid chromatography
NMR: Nuclear magnetic resonance
The processes for preparing Compounds (I) and polyethylene glycol derivatives represented by general formula (II):
X2xe2x80x94CH2(OCH2CH2)nOCH2COxe2x80x94R1xe2x80x94R2xe2x80x94Y2xe2x80x83xe2x80x83(II)
(wherein X2 represents carboxyl, mercapto or 
Y2 represents hydroxyl or 
and R1, R2 and n have the same meanings as defined above) are described below.
Process 1
Process for preparing Compound (Ia), i.e., Compound (I) wherein Z is a group having N, S or O, X1 is CO, and W is a single bond
Compound (Ia) can be prepared according to the following reaction steps. 
(In the formulae, A1 and A2, which may be the same or different, each represents a carboxylic acid protecting group; A3 and A4, which may be the same or different, each represents a carbxylic acid activating group; Hal represents halogen; Z1 represents a group having N, S or O in the definition of Z; and Y1, R1, R2 and n have the same meanings as defined above.)
Examples of the carboxylic acid protecting group are carboxylic acid protecting groups used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen) such as tBu, Bzl, and Pic. An example of the carboxylic acid activating group is ONSu. The halogen means a chlorine atom, a bromine atom or an iodine atom.
(Step 1)
Compound (V) can be obtained by reaction of polyethylene glycol dicarboxylic acid (III) with Compound (IV) in an amount of 0.1 to 1 equivalent, preferably, 0.5 equivalent in a solvent such as DMF in the presence of a base such as potassium carbonate at xe2x88x9250 to 30xc2x0 C. for 1 to 24 hours. Diester and unreactive dicarboxylic acid contained in the obtained product can be removed by partition column chromatography, column chromatography using adsorption resins, reversed-phase silica gel, alumina, diatomaceous earth, or ion-exchange resins, preferably, silica gel column chromatography or thin layer chromatography.
(Step 2)
Compound (VII) can be obtained by condensing Compound (V) with Compound (VI) obtained according to an ordinary liquid-phase peptide synthesis method (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen) in a solvent in the presence of a base in an amount of 1 to 2 equivalents, using a condensing agent in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents. Examples of the base are triethylamine and NMM, examples of the condensing agent are ordinary amino acid condensing reagents such as DCC and EDC, and examples of the solvent are methylene chloride, chloroform and DMF. The reaction is carried out by stirring at 0 to 30xc2x0 C. for 1 to 24 hours.
It is preferred that A2 used as the carboxylic acid protecting group of Compound (VI) is a group which can be selectively removed separately from A1 of Compound (V).
Compound (VII) can also be obtained by condensing Compound (V) with HONSu, HOBt, or the like in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents in a solvent in the presence of an equivalent amount of a base, using a condensing agent in an amount of 1 to 5 equivalents, preferably 1 to 2 equivalents to obtain an active ester, and then by subjecting the obtained ester to reaction with Compound (VI) at 0 to 30xc2x0 C. for 1 to 24 hours. As the base, condensing agent and solvent, those which are described above can be used.
(Step 3)
Compound (VIII) can be obtained by selectively removing the protecting group A2 from Compound (VII) according to a method for the removal of a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
(Step 4)
Compound (X) can be obtained by condensing Compound (VIII) with an equivalent amount of a toxin in a solvent in the presence of a base in an amount of 1 to 2 equivalents, using a condensing agent in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents. Examples of the base are triethylamine and NMM, examples of the condensing agent are ordinary amino acid condensing reagents such as DCC and EDC, and examples of the solvent are methylene chloride, chloroform and DMF. The reaction is carried out by stirring at xe2x88x9230 to 30xc2x0 C. for 1 to 24 hours.
Compound (X) can also be obtained by condensing Compound (VIII) with HONSu, HOBt, or the like in an amount of 1 to 10 equivalents, preferably 1 to 2 equivalents in a solvent in the presence of an equivalent amount of a base, using a condensing agent in an amount of 1 to 5 equivalents, preferably 1 to 2 equivalents to obtain an active ester (IX), and then by subjecting the obtained ester to reaction with a toxin at xe2x88x9230 to 30xc2x0 C. for 1 to 24 hours. As the base, condensing agent and solvent, those which are described above can be used.
(Step 5)
Compound (XI) can be obtained by removing the protecting group A1 from Compound (X) according to a method for the removal of a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen). In the case, for example, where Bzl is used as A1 and tBu as A2, which are to be removed in Steps 3 and 5, respectively, deprotection is carried out according to ordinary methods for selectively removing amino acid protecting groups such as hydration in the presence of a palladium carbon catalyst for A1, and trifluoroacetic acid treatment for A2, whereby A1 and A2 can be selectively removed in each step. It is possible to use the above combination of A1 and A2 in reverse and to reverse the order of deprotection steps.
Compound (XI) can also be obtained by removing the protecting group A1 from Compound (IX) obtained in Step 4 according to the method of Step 5, and then subjecting the obtained compound to reaction with a toxin according to the method of Step 2.
(Step 6)
Compound (Ia) can be obtained from Compound (XI) and a compound which has an affinity for a target cell and has NH, SH, or OH in the molecule according to the method of Step 2. The compounds having an affinity for a target cell, such as proteins and peptides, are liable to be denatured and inactivated in an organic solvent, and it is preferred to carry out the above reaction under mild conditions, e.g. in an aqueous solution. In this case, the reaction is carried out by dissolving a compound having an affinity for a target cell in a buffer such as a phosphate buffer or a borate buffer (pH 6-8), and adding to the solution Compound (XI) in an amount of 1 to 500 equivalents, preferably, 1 to 50 equivalents, and a condensing agent such as EDC, followed by stirring at 0 to 30xc2x0 C. for 1 to 48 hours. Alternatively, the reaction may be carried out by obtaining an active ester (XII) according to the method of Step 2, and adding to a solution of a compound having an affinity for a target cell in a buffer (pH 6-8) the obtained active ester in an amount of 1 to 500 equivalents, preferably 1 to 50 equivalents in the presence of 0 to 10%, preferably 0 to 5% DMSO or DMF, followed by stirring at 0 to 30xc2x0 C. for 1 to 48 hours.
Process 2
Process for preparing Compound (Ib), i.e., Compound (I) wherein Z is a group having N, S or O, X1 is CO, and W is 
Compound (Ib) can be prepared according to the following reaction steps. 
(In the formulae, A1, Y1, Z1, R1, R2 and n have the same meanings as defined above).
(Step 7)
Compound (XIII) can be obtained from Compound (VIII) and aminoethyl maleimide according to the method of Step 2.
(Step 8)
Compound (XIV) can be obtained by subjecting Compound (XIII) to reaction with a toxin. The reaction is carried out by dissolving a toxin in a buffer such as a phosphate buffer and a borate buffer (pH 6-8), and adding Compound (XIII) in an amount of 1 to 50 equivalents to the solution, followed by stirring at 0 to 30xc2x0 C. for 1 to 48 hours.
(Step 9)
Compound (Ib) can be obtained from Compound (XIV) according to the methods of Steps 5 and 6.
Process 3
Process for preparing Compound (Ic), i.e., Compound (I) wherein Z is a group having CO, X1 is S, and W is a single bond
Compound (Ic) can be prepared according to the following reaction steps. 
(In the formulae, A4 represents a thiol protecting group; Z2 represents a group having CO in the definition of Z; and A1, Y1, R1, R2 and n have the same meanings as defined above.)
Examples of the thiol protecting group are benzyl, picolyl, and nitrobenzyl.
The starting compound (XV) can be obtained according to the method for the synthesis of polyethylene glycol derivatives described in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed., Plenum, NY. 1992).
(Step 10)
Compound (XVI) can be obtained by protecting the thiol group of Compound (XV) according to a method for introducing a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
(Step 11)
Compound (XVII) can be obtained from Compound (XVI) according to the method of Step 3.
(Step 12)
Compound (XVIII) can be obtained from Compound (XVII) according to the methods of Steps 2 and 3.
(Step 13)
Compound (XIX) can be obtained from Compound (XVIII) according to the method of Step 4.
(Step 14)
Compound (XX) can be obtained by deprotecting Compound (XIX) according to a method for removing a protecting group used in ordinary peptide synthesis (Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya et al., Maruzen).
(Step 15)
Compound (Ic) can be obtained by binding Compound (XX) to a compound having an affinity for a target cell and having COOH in the molecule by a method such as the activation of a thiol group described in J. Applied Biochem., 6, 56-63 (1984).
Process 4
Process for preparing Compound (Id), i.e., Compound (I) wherein Z is a group having N, S or O, X1 is 
xe2x80x83and W is a single bond, and Compound (IId), i.e., Compound (II) wherein X2 is 
xe2x80x83and Y2 is hydroxyl.
Compound (Id) and Compound (IId) can be prepared according to the following reaction steps. 
(In the formulae, A1, Y1, Z1, R1, R2 and n have the same meanings as defined above.)
The starting compound (XXI) can be obtained according to the method for the synthesis of polyethylene glycol derivatives described in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed., Plenum, NY. 1992).
(Step 16)
Compound (XXII) can be obtained from Compound (XXI) according to the method of Step 3.
(Step 17)
Compound (IId) can be obtained from Compound (XXII) according to the methods of Steps 2 and 3.
(Step 18)
Compound (XXIII) can be obtained from Compound (IId) according to the method of Step 4.
(Step 19)
Compound (Id) can be obtained from Compound (XXIII) and a compound having an affinity for a target cell and having NH, SH or OH in the molecule according to the method of Step 8.
Process 5
Process for preparing Compound (IIa), i.e., Compound (II) wherein X2 is carboxyl and Y2 is hydroxyl
(Step 20)
Compound (IIa) can be obtained from Compound (VIII) according to the method of Step 3.
Process 6
Process for preparing Compound (IIb), i.e., Compound (II) wherein X2 is carboxyl and Y2 is 
(Step 21)
Compound (IIb) can be obtained from Compound (XIII) according to the method of Step 3.
Process 7
Process for preparing Compound (IIc), i.e., Compound (II) wherein X2 is mercapto and Y2 is hydroxyl
(Step 22)
Compound (IIc) can be obtained from Compound (XVIII) according to the method of Step 14.
Compounds (I) and (II) having the desired groups at the desired positions can be obtained by combining the above-described methods appropriately.
The intermediates and desired compounds in the above-described processes can be isolated and purified by purification methods such as filtration, extraction, washing, drying, concentration, recrystallization, various kinds of column chromatography, e.g. silica gel chromatography, ion-exchange chromatography, reversed-phase chromatography, and gel filtration chromatography, and dialysis using an ordinary semipermeable membrane. The intermediates can be subjected to the subsequent reaction without a specific purification treatment.
Examples of Toxin Conjugates (I) obtained by the above-described processes are shown in Table 1.
The pharmacological activities of the toxin conjugates are shown below by Test Examples.
The inhibitory effect of the toxin conjugates on cell growth was examined. Cervical cancer HeLaS3 cells (CALLAxe2x88x92) having no expression of CALLA antigen and Burkitt lymphoma Daudi cells (CALLA+) having an expression of CALLA antigen were used as target cell lines. Each of the target cell suspensions was put into wells of a 96-well flat plate in an amount of 50 xcexcl (1xc3x97103 cells/well), and cultured in a CO2-incubator at 37xc2x0 C. for 2 hours. After culturing, various dilutions of a toxin conjugate or a monoclonal antibody were respectively added in an amount of 50 xcexcl, followed by further culturing in the CO2-incubator at 37xc2x0 C. for 68 hours. Then, 20 xcexcl of 3H-thymidine (463 KBq/ml) was added to each well, and after 4 hours, the cells were harvested to determine the radioactivity of 3H-thymidine incorporated into the cells by using Matrix 96 (Packard Japan). The cell growth inhibiting activity was calculated according to the following equation.       (          1      -                        Radioactivity of treated cells                          Radioactivity of control cells                      )    xc3x97  100
As a result, Compound (Ia-3) and Compound (Ia-1) exhibited a little inhibitory effect on the growth of HeLaS3 cells at high concentrations, whereas they exhibited a remarkable inhibitory effect on the growth of Daudi cells even at very low concentrations. When Compound (Ia-9) or Compound (Ia-7) was added, an inhibitory effect was hardly observed on the growth of HeLaS3 cells, while a more specific inhibitory effect was observed on the growth of Daudi cells. Addition of the monoclonal antibody (NL-1) alone had little effect on the cell growth (refer to FIG. 1).
The inhibitory effect of Compound (Ia-6) on cell growth was examined in the same manner as in Test Example 1. Cervical cancer HeLaS3 cells (sLeaxe2x88x92) having no expression of sLea antigen and large intestine cancer SW1116 cells (sLea+) having an expression of sLea antigen were used as target cell lines. As a result, an inhibitory effect was observed on the growth of SW1116 cells, but not on the growth of HeLaS3 cells. When a monoclonal antibody (KM-231) alone was added, no cell growth inhibiting effect was observed on either of these strains. The antigen-specific cell growth inhibiting effect of the conjugate was thus confirmed (refer to Table 2).
From the foregoing, the antigen-specific cell growth inhibiting effect of various kinds of conjugates and the utility of the spacers were confirmed.
The inhibitory effect of Compound (Ia-10) on cell growth was examined. Cervical cancer HeLaS3 cells (sLeaxe2x88x92) having no expression of sLea antigen and large intestine cancer SW1116 cells (sLea+) having an expression of sLea antigen were used as target cell lines. Each of the target cell suspensions was put into wells of a 96-well flat plate in an amount of 50 xcexcl (1xc3x97103 cells/well), and cultured in a CO2-incubator at 37xc2x0 C. for 2 hours. After culturing, various dilutions of the drug-monoclonal antibody conjugate or a monoclonal antibody were respectively added in an amount of 50 xcexcl, followed by further culturing in the CO2-incubator at 37xc2x0 C. for 2 hours. Then, the cells in the plate were centrifuged, and immediately after removal of the supernatant, 100 xcexcl of a medium was added, followed by further culturing for 64 hours. To each well was added 20 xcexcl of 3H-thymidine (463 KBq/ml), and after 4 hours, the cells were harvested to determine the radioactivity of 3H-thymidine incorporated into the cells by using Matrix 96 (Packard Japan). The cell growth inhibiting activity was calculated according to the following equation.       (          1      -                        Radioactivity of treated cells                          Radioactivity of control cells                      )    xc3x97  100
As a result, Compound (Ia-10) exhibited an inhibitory effect on the growth of SW1116 cells, but no effect on the growth of HeLaS3 cells. When the monoclonal antibody (KM-231) alone was added, an inhibitory effect was slightly observed only on the growth of SA1116 cells. The antigen-specific cell growth inhibiting effect of the conjugate and the utility of the spacer were thus confirmed also in this assay system (refer to Table 3).
Human myeloma SK-Ly-18 cells were suspended in RPMI-1640 medium containing 10% fetal calf serum at a concentration of 2xc3x97108 cells/ml, and the suspension was mixed with Matrigel (registered trademark; Becton Dickinson Labware, USA) in the ratio of 1:1 (v/v). The mixture (0.1 ml, 1xc3x97107 cells/mouse) was subcutaneously transplanted into BALB/C nu/nu mice (Clea Japan, Inc.). On the 7th day after the tumor transplantation, a drug-monoclonal antibody conjugate (amount corresponding to 7.5 mg/kg ADM) or ADM (7.5 mg/kg) was intravenously administered to the mice divided in groups each consisting of 5. A control group was given physiological saline in the same manner. The major axis and the minor axis of tumor were measured at intervals, and the tumor volume was calculated as an approximation value of an ellipsoid according to the following equation.
Tumor Volume (mm3)=(axc3x97b2)/2
a: major axis (mm) b: minor axis (mm)
The therapeutic effect on the transplanted tumor was evaluated in terms of V/V0, the ratio of the tumor volume on the day of evaluation (V) to that on the day of drug administration (V0).
As a result, a significant tumor growth was observed in the control group, and a remarkable tumor growth inhibiting effect was observed in Compound (Ia1)- and Compound (Ia-3)-administered groups. On the other hand, a growth inhibiting effect was not observed in the test group to which the same quantity of ADM alone was given compared with the control group. The utility of the drug-monoclonal antibody conjugates was thus demonstrated (refer to FIG. 2).